1. Field of the Invention
The present invention is related to HIV therapy and prophylaxis. In particular, the invention relates to methods for eliciting broadly neutralizing antibodies that target entry-relevant structures of HIV-1 gp41. Such methods, and pharmaceutical compositions therefor, can be employed to inhibit HIV entry into uninfected cells.
2. Related Art
The development of effective vaccines to prevent infection with HIV remains a high priority goal. To date, envelope glycoproteins (gp160 and gp120/gp41) have been the main focus of vaccine research efforts. One result of this work is the observation that the humoral response generated against native forms of the envelope (primarily oligomeric forms of the gp120/gp41 complex) is more broadly neutralizing than antibody raised against denatured and/or monomeric envelope (VanCott, T. C., et al., J. Virol. 71:4319-4330 (1997)). Structural considerations are important components for both understanding the immunogenicity of the envelope protein and the design of envelope based immunogens which induce a broad neutralizing response against HIV.
A good deal of structural information is available with respect to the transmembrane protein (TM or gp41). Predictive work indicated that several regions of the ectodomain of gp41 display a high propensity to exhibit certain specific types of secondary structure (Gallaher, W. R., et al., AIDS Res. Hum. Retroviruses 5:431-440 (1989); Delwart, E. L., et al., AIDS Res. Hum. Retroviruses 6:703-704 (1990)). Experimental work employing both synthetic peptides and protein recombinants has established that these predictions were generally correct and recently a three dimensional structure for a portion of the gp41 ectodomain was reported (Wild, C., et al., Proc. Natl. Acad. Sci. USA 89:10537-10541 (1992); Wild, C., et al., Proc. Natl. Acad. Sci. USA 91:12676-12680 (1994); Wild, C., et al., AIDS Res. Hum. Retroviruses 11:323-325 (1995); Chan, D. C., et al, Cell 89:263-273 (1997)). Results from both solution studies and crystallographic analysis indicate that in one form this structured region of the transmembrane protein is a primer of two interacting regions of gp41. This trimeric structure is a six helix bundle consisting of an interior parallel coiled-coil primer (region one) which associates with three identical α-helices (region two) which pack in an oblique, antiparallel manner into the hydrophobic grooves on the surface of the coiled-coil trimer (FIG. 3). This hydrophobic self-assembly domain is believed to constitute the core structure of gp41.
A series of studies carried out using both synthetic peptides and recombinant proteins modeling the distal regions of the TM involved in generating this structure suggest that it (or the gp41 regions from which it is derived) plays a critical role in the process of HIV-1 entry (Wild, C., et al., Proc. Natl. Acad. Sci. USA 89:10537-10541 (1992); Wild, C., et al., AIDS Res. Hum. Retroviruses 9:1051-1053 (1993);Wild, C., et al., Proc. Natl. Acad. Sci. USA 91:12676-12680 (1994); Wild, C., et al., AIDS Res. Hum. Retroviruses 11:323-325 (1995); Wild, C., et al., Proc. Natl. Acad. Sci. USA 91:9770-9774 (1994); Chen, C. -H., et al., J. Virol. 69:3771-3777 (1995)).
The functional role of the transmembrane protein of HIV-1 in virus replication was shown when the region of the ectodomain of the TM corresponding to amino acid residues 558-595, which was predictive of α-helical secondary structure (Gallaher, W. R., et al., AIDS Res. Hum. Retroviruses 5:431-440 (1989); Delwart, E. L., et al., AIDS Res. Hum. Retroviruses 6:703-704 (1990)), formed a coiled-coil structure when modeled as a synthetic peptide (Wild, C., et al., Proc. Natl. Acad. Sci. USA 89:10537-10541 (1992)). The peptide modeling this region, DP-107, was shown to be a potent, virus specific inhibitor of HIV replication and the inhibitory activity was related to the structural components exhibited by the peptide. In both neutralization and cell-cell fusion assays, the DP-107 peptide completely blocked virus infection at concentrations of 1.0 μg/ml. Unlike other inhibitors of HIV replication (i.e. soluble CD4) and most neutralizing sera, the activity of the DP-107 peptide was not isolate restricted. Using a series of DP-107 analogs containing structure disrupting point mutations and a set of HIV-1 envelope constructs containing identical mutations, it has been shown that the structural components of the coiled-coil region of the TM were critical to both virus entry and fusion phenotype and that mutations which disrupted this gp41 structure gave rise to an envelope complex which was unable to mediate virus entry (Wild, C., et al., Proc. Natl. Acad. Sci. USA 91:12676-12680 (1994)).
Studies of the coiled-coil domain of gp41 resulted in the identification of a second region of the ectodomain of the TM, which when modeled as a synthetic peptide, was also a potent, virus specific inhibitor of HIV replication (Wild, C., et al., AIDS Res. Hum. Retroviruses 9:1051-1053 (1993)). However, unlike the DP-107 region, the peptide corresponding to amino acid residues 643-678 of the TM (DP-178), did not exhibit stable solution structure. Experiments with the DP-107 and DP-178 peptides established that both of these materials blocked HIV replication at an early step, most likely during virus entry (Wild, C., et al., Proc. Natl. Acad. Sci. USA 91:9770-9774 (1994)). This observation led to speculation that these peptides might inhibit virus replication by interacting with and disrupting determinants within the TM that were critical for virus entry. Efforts to better define the higher order structural components that were present in gp41 and functioned during virus entry led to the observation that the distal regions of the TM modeled by the two inhibitory peptides (DP-107 and DP-178) did interact with one another to form an oligomeric structure (Wild, C., et al., AIDS Res. Hum. Retroviruses 11:323-325 (1995); Chen, C. -H., et al., J. Virol. 69:3771-3777 (1995)). Recently, this oligomeric structure was characterized as a trimeric, six helix bundle consisting of an interior parallel coiled-coil trimer (DP-107 region) which associates with three identical α-helices (DP-178 region) which pack into the hydrophobic grooves on the surface of the coiled-coil trimer (FIG. 3) (Chan, D. C., et al., Cell 89:263-273 (1997)).
Research has focused on determining the functional role of these gp41 structural determinants in virus entry. DP-107 and DP-178 peptides interact in a specific manner with the ectodomain of gp41 and this interaction is critical to their inhibitory activities.
U.S. Pat. No. 5,464,933, Bolognesi et al., describes peptides which exhibit potent anti-retroviral activity. Specifically disclosed are the peptide DP-178 (SEQ ID NO:3) derived from the HIV-1LAI gp41 protein, as well as fragments, analogs and homologs of DP-178. The peptides are used as direct inhibitors of human and non-human retroviral transmission to uninfected cells. The patent teaches that the peptides may also be prophylactically employed in individuals after such individuals have had an acute exposure to HIV.
U.S. Pat. No. 5,656,410, Wild et al., describes protein fragments derived from the HIV transmembrane glycoprotein (gp41), including the peptide DP-107 (SEQ. ID NO:1) which have antiviral activity. Also disclosed are methods for inhibiting enveloped viral infection, and methods that modulate biochemical processes involving coiled coil peptide interactions.
While recent work has increased knowledge of the structural components of the HIV-1 transmembrane protein, the immunogenic nature of gp41 remains poorly understood. It is known that one of two immunodominant regions present in the HIV-1 envelope complex is located in gp41 (Xu, J. -Y., et al., J. Virol. 65:4832-4838 (1991)). This determinant (TM residues 597-613) is associated with a strong, albeit non-neutralizing humoral response in a large number of HIV+ individuals. Also, the broadly neutralizing antibody, 2F5, maps to the ectodomain of gp41 (TM residues 662-667) (Muster, T., et al., J. Virol. 67:6642-6647 (1993); Muster, T., et al., J. Virol. 68:4031-4034 (1994)). It is interesting to note that this antibody maps to a determinant of the TM that overlaps one of the two regions of gp41 which interact to form the recently characterized hydrophobic core of the protein (FIG. 1). This observation has lead to speculation that 2F5 might actually neutralize virus by interacting with and disrupting the function of an entry-relevant gp41 structure. An extensive study which mapped the antigenic structure of gp41 supports this idea. This work characterized several conformation dependent gp41 MAbs which mapped to the same region of the TM as 2F5 (Earl, P. L., et al., J. Virol. 71:2647-2684 (1997)). Although the binding sites for these non-neutralizing monoclonal antibodies (MAbs) overlapped the 2F5 determinant, in competition experiments neither of these antibodies was blocked from binding to native protein by the 2F5 MAb. This indicates that while the two dimensional regions to which these antibodies map are similar, the three dimensional epitopes to which they bind are quite different.
The observation that only one neutralizing MAb (2F5) maps to the ectodomain of gp41 and that antibodies to the 2F5 epitope are poorly represented in sera from HIV infected individuals suggests that, for the most part, gp41 neutralizing epitopes are cryptic. The cryptic nature of these neutralizing epitopes is most likely related to the functional role of the TM in HIV-1 replication which involves mediating virus entry. It has been shown that prior to gp120-CD4 binding the HIV envelope complex exists in a non-fusogenic form. While the exact nature of this pre-entry form is unknown, binding experiments have established that the non-fusogenic state is characterized by the inaccessibility of large portions of the gp41 ectodomain (Sattentau, Q. J. and J. P. Moore, J. Exp. Med. 174:407-415 (1991); Sattentau, Q. J., et al., Virol. 206:713-717 (1995)). However, once binding of virus to target cell has occurred, the gp120-gp41 complex undergoes a series of conformational changes that involve reorganization of both the extracellular surface component of the HIV-1 envelope protein (SU or gp120) and TM proteins and the formation of structural components within the TM which are believed to be critical to virus entry. Although the steps involved in the transition from the non-fusogenic to fusogenic state are largely unknown, it is believed that this transformation is characterized by the formation of a series of structural intermediates within the transmembrane protein which drive the conformational changes required for virus entry. The transitory nature of this event and the structures associated with it, rather than the absence of appropriate structural determinants, are believed to account for the poor neutralizing response to the TM component of the envelope system.
Attention has been given to the development of vaccines for the treatment of HIV infection. The HIV-1 envelope proteins (gp160, gp120, gp41) have been shown to be the major antigens for anti-HIV antibodies present in AIDS patients (Barin, et al., Science 228:1094-1096 (1985)). Thus far, these proteins seem to be the most promising candidates to act as antigens for anti-HIV vaccine development. To this end, several groups have begun to use various portions of gp160, gp120, and/or gp41 as immunogenic targets for the host immune system. However, prior art attempts have thus far met with minimal success.
Thus, although a great deal of effort is being directed to the design and testing of HIV vaccines, an effective vaccine is needed.